xxxii Scientific Proceedings. XVII
made in different ways, the identity of the active substance
cannot he regarded as established. The possibility remains that
the protein described contains the active material as admixture.
The method of preparation of solutions suitable for experimental
workwhichwe have found the most satisfactory is briefly as follows:
For each kilo of finely ground fresh pancreas hash add about
40 cc. of 10 N H2S04 (or 40 cc. concentrated HC1), 1,200 cc. of
95 per cent alcohol, and 300 cc. of water. Stir well at intervals
during 4 to 12 hours; at room temperature. Strain through cloth
and press out liquid as completely as possible. Reextract hash
with a liter or more of 60 per cent alcohol, and again strain and
press out liquid. To the combined very turbid liquid add NaOH
to faint acid reaction to litmus. Filter through paper, and evapo-
rate filtrate placed in shallow trays in a warm air current (tempera-
ture of solution 20 to 30°C.) until all odor of alcohol is gonc, or
until the volume is reduced to one-quarter to one-tenth the original
volume. Transfer the liquid without filtering to a separatory
funnel, rinsing the dishes with small amounts of water. Acidify
slightly with HC1 or H2S04, add 40 gm. of solid (NH4)2SO4 for
each 100 cc. of solution and shake until dissolved. On standing a
few hours the flocculent precipitate first formed rises to the sur-
face and coagulates to a compact layer. Separate the lower
liquid as completely as possible, leaving the precipitate in the
funnel to drain. When all possible liquid has drained off, add
to funnel 75 per cent alcohol (about 50 cc. for each kilo of pan-
creas hash used), which dissolves the insulin. Rinse the solu-
tion and insoluble residue into a centrifuge tube with small
amounts of 60 per cent alcohol, and centrifugate. Pipette off
the clear alcohol solution and add 8 to 10 volumes of 95 per cent
or absolute alcohol. Adjust the reaction of the mixture to C,
5 to 6 and filter after standing some hours. The precipitate is
dissolved in water with the addition of small amounts of either
acid or alkali. We ordinarily obtain one "1 kg. rabbit unit"
from each 2 gm. of pancreas hash used. Occasionally a better
yield has been obtained. For use in human diabetes the material
should be purified by means of repeated precipitation at the
isoeleotric point, and by precipitation by ammonium sulfate, and
by alcohol.